Development of clade- (Roseobacter and Alteromonas) and taxon-specific oligonucleotide probes to study interactions between toxic dinoflagellates and their associated bacteria

Bacteria associated with toxic algae in culture have been implicated in the enhancement of algal toxin production and auto-toxigenesis. Toxigenic and non-toxigenic bacterial isolates from Alexandrium spp. have been determined to belong predominantly to the alpha- and gamma- subclasses of the class Proteobacteria. Within these subclasses, the isolates were further classified into two clades that were phylogenetically affiliated with (1) the genus Roseobacter. a newly identified group that is of major significance within the alpha -Proteobacteria and (2) the genus Alteromonas. Specific 16S rRNA sequence signatures were identified for these clades as well as for three pairs of isolates and for one subcluster within the Roseobacter clade and for three clusters representing eight isolates in the Alteromonas clade. Oligonucleotide probes complementary to these regions were designed and their specificities were tested using dot-blot and whole-cell in situ hybridizations of target and non-target strains. The empirically determined dissociation temperatures of the probes ranged between 55 and 65 making them applicable as a set for screening environmental samples. In situ hybridization of bacteria in cultures of Alexandrium spp. with the clade-specific probes showed a loose association of these bacteria within the phycosphere. in addition, the fluorescent signal of the probes was bright and easily distinguishable from autofluorescent bacteria and the dinoflagellates.